Human steroid receptor

ABSTRACT

Through the use of the novel receptor NER in a screening procedure, TOFA (5-tetradecyloxy)-2-furan-carboxylic acid) has been found to modulate other receptors and to be a potent potentiator of other drugs. TOFA activates the NER receptor. The NER receptor is a novel member of the steroid hormone receptor family and has been prepared by cDNA cloning from a human osteosarcoma SAOS-2/B10 cell library. Also disclosed is the complete sequence of human NER cDNA; a COS stable expression system; the expressed NER protein; and an assay using the COS expression system. In addition, the invention relates to a method for identifying functional ligands of the NER receptor.

This application is the U.S. national phase of PCT/US95/13924, filedOct. 24, 1995, which is a continuation of U.S. patent application Ser.No. 08/330,283, filed Oct. 27, 1994, now U.S. Pat. No. 5,679,518.

SUMMARY OF THE INVENTION

The present invention relates generally to a method of findingpotentiators of receptors employing a screening procedure using thenovel recombinant human steroid hormone receptor hereinafter called NER.The compound TOFA (5-(tetradecyloxy)-2-furan carboxylic acid) has beenfound through the above screening procedure employing NER to be apotentiator of ligands for other receptors, particularly G-proteincoupled receptors, without having any independent effect on thereceptors.

Compounds which activate the NER receptor, such as TOFA, potentiate theeffects of nerve growth factor (NGF) and may be useful in the treatmentof Alzheimer's disease. These compounds may also be useful inpotentiating the effects of muscarinic agonists in the treatment ofocular hypertension. Further, compounds which activate the NER receptorare also useful in potentiating the effects of dopamine D1 antagonistsin the treatment of psychoses, particularly schizophrenia, and in thetreatment of movement disorders such as distonia, tardive dyskinesia andGilles de la Tourette syndrome. Further NER activators may potentiatethe prevention of the development of intraocular pressure induced bydopamine agonists in hydrodynamic disorders of the eye and in patientswith increased intracranial pressure.

The novel recombinant steroid hormone receptor NER has been prepared bypolymerase chain reaction techniques. Also disclosed are the completesequence of human NER complementary DNA; expression systems, including aCOS-stable expression system; and an assay using the COS expressionsystem. In addition, the invention relates to a method for identifyingfunctional ligands of the NER receptor.

BACKGROUND OF THE INVENTION

Retinoids, steroid and thyroid hormones and possibly other moleculesproduce their biological effects by binding to proteins of the steroidreceptor superfamily. These receptors interact with specific DNAsequences and modulate gene expression (for reviews see J M Berg, Cell57:1065-1068 (1989); R M Evans, Science 240:899-895 (1988); M Beato,Cell 56:335-344 (1989)). Sequence analysis and functional studies ofthese receptors revealed two important regions which exhibit a highdegree of amino acid residue conservation. The highest level ofsimilarity among the receptors is found in a region which contains ninecysteine residues that bind zinc atoms to form two "zinc fingers," whichinteract with the cognate steroid response elements of DNA (J Miller, etal., EMBO J 4:1609-1614 (1985); R M Evans, Cell 52:1-3 (1988)). Thesecond region, which is less conserved, is the ligand-binding domain,responsible for the interaction with the hormone (J. Carlstedt-Duke, etal., Proc Natl Acad Sci USA 79:4260-4264 (1982). J. Carlstedt-Duke, etal., Proc Natl Acad Sci USA 84:4437-4440 (1987)). Recent studies haveattributed additional functions to other domains of these receptors,such as protein-protein interaction that participates in transcriptionalregulation (R Scule, et al., Cell 62:1217-1226 (1990); H F Yang, Cell62:1205-1215 (1990); J M Holloway et al., Proc Natl Acad Sci USA87:8160-8164 (1990)). The amino acid conservation in the DNA bindingdomain has led to the identification of new members of the steroidreceptor superfamily.

For example, hER1 and hER2 have been cloned by low stringencyhybridization of cDNA libraries with a DNA probe coding for the DNAbinding domain of the estrogen receptor (Giguere, et al., Nature331:91-94 (1988)). Similar approaches have led to the discovery of theretinoic acid receptors and the peroxisome proliferator activatorreceptor (PPAR) (I Issemann, et al., Nature 347:645-650 (1990); D JMangelsdorf, et al., Nature 345:224-229 (1990)). Recently, three novelmembers of the Xenopus nuclear hormone receptor superfamily have beendisclosed (C Dreyer, Cell 68:879-987 (1992)). In addition, U.S. Pat. No.4,981,784 to Evans, et al. discloses the identification of a retinoicacid receptor and the use of chimeric constructs to produce hybridreceptors for the identification of novel ligands. The above references,however, neither disclose nor suggest the instant invention.

TOFA (5-(tetradecyloxy)-2-furan-carboxylic acid) has been reported toinhibit fatty acid synthesis by inhibiting acetyl-CoA carboxylase, therate limiting step in de novo fatty acid synthesis, in vivo ##STR1## athigh doses, i.e. 0.15% of diet . (See, e.g. Arbeeny, "Inhibition offatty acid synthesis decreases renal low density lipoprotein secretionin the hamster," J. Lipid Res. 33: 843-851, (1992); Ribeneau-Gyon andGilles, FEBS Lett. 62: 309-312, (1976); Halvorson and McCune,"Inhibition of fatty acid synthesis in isolated adipocytes by5-tetradecyloxy)-2-furoric acid," Lipids 19(11): 851-856, (1984); Ottoet al., "Reciprocal effects of 5-(tetradecyloxy)-2-fuoric acid on fattyacid oxidation," Arch. Biochem. Biophys. 242(1):23-31, (1985); Parker etal., "5-(tetradecyloxy)-2-furancarboxylic acid and related hypolipidemicfatty acid-like alkyloxyarylcarboxylic acids," J. Med. Chem. 20:781-791,(1977)). The present invention comprises in one embodiment the use oflow-dose TOFA to potentiate the activity of ligands of G-coupledreceptors and to potentiate the activity of endogenously producedhormones or neurotransmitters.

Powell et al., ("Dopamine activation of an orphan of the steroidreceptor family" Science 252: 1546-48, (1991), and "Dopaminergic andligand independent activation of steroid hormone receptors" Science 254:1636-39, (1991)) have reported that dopamine activates transcription,mediated by steroid hormone receptors, by a ligand-independentmechanism. However, unlike dopamine, TOFA and activators of NER do notthemselves have dopaminergic activity at the dosage level used.

Dopamine receptors are membrane proteins that have seven transmembranedomains and mediate transmembrane signaling via the heterotrimeric Gproteins. The receptors are predominantly localized in the centralnervous system, but peripheral organs such as the kidney, loweresophagus and cardiac and mesenteric arteries also respond to dopaminethrough specific binding sites. (Strange, "Dopamine receptors: structureand function," Prog Brain Res. 99:167-79, (1993); Strange, "New insightsinto dopamine receptors in the central nervous system," Neurochem. Int.22(3):223-236, (1993).) Molecular cloning revealed that this receptorfamily consists of five genes, D1-D5, that modulate the activity ofadenyl cyclase. The D1 and D5 dopamine receptors stimulate adenylylcyclase activity, while the D2, D3 and D4 receptors inhibit this enzyme.(Seeman and Van-Tol, "Dopamine receptor pharmnacology," Curr. Opin.Neurol. Neurosurg. 6(4):602-609, (1993); Kebabian, "Multiple dopaminereceptors and their implications in medicine," Curr. Opin. Neurol.Neurosurg. 5(4): 514-518, (1992); Kebabian, "Brain dopamine receptors:20 years of progress," Neurochem. Res. 18(1):101-104, (1993); Sibley, etal., "Molecular neurobiology of dopaminergic receptors," Int. Rev.Neurobiol. 35:391-415, (1993).)

Dopaminergic agents and their antagonists serve to treat movementdisorders, other neuropsychiatric disorders, nausea, and certainhormonal disorders.

Dopamine D₁ receptors are coupled to adenyl cyclase. Known Dopamine D₁antagonists include SCH23390(8-chloro-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-3-benzazepin-7-olhemimaleate): ##STR2## Dopamine D1 antagonists have demonstrated a rolein the treatment of Alzheimer's disease. In the radial-arm maze test, anassay used for testing memory enhancing agents, medial cholinergicpathway lesions produce evidence of memory loss and chronic treatmentwith SCH23390 reversed the lesion-induced impaired performance. (McGurket al. "Dopaminergic drugs reverse the impairment of radial-arm mazeperformance caused by lesions involving the cholinergic medial pathway,"Neuroscience. 50(1):129-135, (1992)).

Dopamine D1 receptor antagonists are also useful in the treatment ofmovement disorders such as Gilles de la Tourette syndrome, dystonia, andtardive dyskinesia. Dopamine D1 receptor antagonists are also useful intreating psychoses, most particularly schizophrenia.

Gilles de la Tourette syndrome, or hereditary multiple tic disorder,begins in childhood with simple tics but progresses to multiple, complexmovements including respiratory and vocal tics. In 50% of patients,coprolalia, involuntary scatologic utterances, occurs. The tics andcoprolalia may be severe enough to be physically and socially disabling.Gilles de la Tourette syndrome is generally treated with haloperidol,0.5 to 40 mg/day. The dosage of haloperidol is limited by side effectssuch as dysphonia, parkinsonism and akathisia. Clonidine, 0.1 to 0.6mg/day may also be effective in some patients, but is limited by theside effect of hypotension. The present invention, by potentiating thedopamine D1 receptor antagonists, permits the treatment of Gilles de laTourette syndrome with a decreased administration of dopamine D1receptor antagonists.

Dystonia is characterized by sustained abnormal postures and disruptionsof ongoing movement resulting from alterations in muscle tone. Dystoniais generally treated with high dose anticholinergics such astrihexyphenidyl 6 to 30 mg/day, benztropine 3 to 14 mg/day, andreserpine, a dopamine depleting drug, 0.1 to 0.6 mg/day.

Tardive dyskinesia is characterized by choreiform movements of thebuccal-lingual-fascial muscles, less commonly the extremities. Rarely,focal or even generalized dystonia may be seen. Tardive dyskinesia maybe caused by high doses of phenothiazines given over a long time, acommon practice in young schizophrenics. Older patients, particularlywomen and those with brain injury, have a higher incidence of tardivedyskinesia. The problem does not disappear when the drug is discontinuedand resists standard treatments for movement disorders. Anticholinergicscan exascerbate tardive dyskinesia. The incidence has increased with thecommon and prolonged use of phenothiazines. By potentiation the effectof dopamine D1 receptor antagonists administered, NER receptoractivators such as TOFA, are useful in the prevention of tardivedyskinesia.

Known dopamine D1 receptor agonists include SKF 38390(1-phenyl-7,8-dihydroxy-2,3,4,5-tetrahydro-1H-3-benzazepine): ##STR3##

Dopamine D₁ receptor agonists are useful in treating Parkinson'sDisease.

Haloperidol is a preferential blocker of dopamine D2 receptors. OtherDopamine D₂ receptor modulators include dihydroergocryptine andbromocryptine. ##STR4##

The muscarinic receptors naturally bind acetylcholine. ##STR5## Theplant alkaloid muscarine also activates the muscarinic cholinergicreceptors. ##STR6##

Muscarinic receptors occur at post ganglionic parasympathetic terminalsinvolved in gastrointestinal and ureteral peristalsis, the promotion ofglandular secretion, pupillary constriction, peripheral vasodilation andreduction in heart rate. The muscarinic receptor is also a G-proteincoupled receptor, and its stimulation causes an increase in cGMP.Pilocarpine is a known muscarinic agonist. ##STR7## Others includecarbachol, metacholine, betanechol, arecoline, and oxotremorine.##STR8## Muscarinic agonists are useful for the treatment of ocularhypertension, particularly in glaucoma, and to stimulate thegastrointestinal tract and urinary bladder to relieve post operativeatony.

NGF, nerve growth factor, is required for the development of sympatheticand sensory neurons and for neuronal viability in mature brain cells.NGF treatment induces the expression of the immediate early responsegene--the orphan steroid hormone receptor Nur77. (Davis et al.,"Transcriptional activation by Nur77, a growth factor inducible memberof the steroid hormone receptor superfamily. Mol Endocrinol. 5(6):854-859, 1991; Hazel et al., "Nur77 is differentially modified in PC12cells upon membrane depolarization and growth factor treatment," MolCell Biol 11(6):3293-3246, 1991; Milbrandt, "Nerve growth factor inducesa gene homologous to the glucocorticoid receptor gene," Neuron1(3):183-188, 1993). Due to its role in neuronal maintenance and itsability to stimulate nerve growth, NGF is potentially important for thetreatment of Alzheimer's disease. (Olson, "Reparative strategies inbrain-treatment strategies based on trophic factors and cell transfertechniques," Acta Neurochirurgica 58:3-7, 1993). The potential benefitof NGF in Alzheimer's disease is also suggested by the recentdemonstration of memory improvement following intracranial infusion ofNGF in an Alzheimer's patient. Thus, NGF appears to be able tocounteract the cholinergic deficits of Alzheimer's disease. (Seiger etal. "Intercranial infusion of purified nerve growth factor to analzheimer patient," Behavioral Brain Research 57:255-261, 1993).

DETAILED DESCRIPTION OF THE INVENTION

One embodiment of the invention concerns human steroid hormone receptorNER, said receptor being free of other human receptor proteins.

In one class this embodiment concerns human steroid hormone receptorNER, said receptor being free of other human proteins.

Within this class, this embodiment concerns human steroid hormonereceptor NER from human cells such as osteosarcoma, said receptor beingfree of other human proteins.

In a second class, this embodiment concerns a protein comprising thefollowing 461 amino acid sequence (SEQ ID NO:2:) depicted from the aminoto the carboxy terminus:

    Met Ser Ser Pro Thr Thr Ser Ser Leu Asp Thr Pro Leu Pro Gly Asn    1               5                   10                  15    Gly Pro Pro Gln Pro Gly Ala Pro Ser Ser Ser Pro Thr Val Lys Glu                20                  25                  30    Glu Gly Pro Glu Pro Trp Pro Gly Gly Pro Asp Pro Asp Val Pro Gly            35                  40                  45    Thr Asp Glu Ala Ser Ser Ala Cys Ser Thr Asp Trp Val Ile Pro Asp        50                  55                  60    Pro Glu Glu Glu Pro Glu Arg Lys Arg Lys Lys Gly Pro Ala Pro Lys    65                  70                  75                  80    Met Leu Gly His Glu Leu Cys Arg Val Cys Gly Asp Lys Ala Ser Gly                    85                  90                  95    Phe His Tyr Asn Val Leu Ser Cys Glu Gly Cys Lys Gly Phe Phe Arg                100                 105                 110    Arg Ser Val Val Arg Gly Gly Ala Arg Arg Tyr Ala Cys Arg Gly Gly            115                 120                 125    Gly Thr Cys Gln Met Asp Ala Phe Met Arg Arg Lys Cys Gln Gln Cys        130                 135                 140    Arg Leu Arg Lys Cys Lys Glu Ala Gly Met Arg Glu Gln Cys Val Leu    145                 150                 155                 160    Ser Glu Glu Gln Ile Arg Lys Lys Lys Ile Arg Lys Gln Gln Gln Gln                    165                 170                 175    Glu Ser Gln Ser Gln Ser Gln Ser Pro Val Gly Pro Gln Gly Ser Ser                180                 185                 190    Ser Ser Ala Ser Gly Pro Gly Ala Ser Pro Gly Gly Ser Glu Ala Gly            195                 200                 205    Ser Gln Gly Ser Gly Glu Gly Glu Gly Val Gln Leu Thr Ala Ala Gln        210                 215                 220    Glu Leu Met Ile Gln Gln Leu Val Ala Ala Gln Leu Gln Cys Asn Lys    225                 230                 235                 240    Arg Ser Phe Ser Asp Gln Pro Lys Val Thr Pro Trp Pro Leu Gly Ala                    245                 250                 255    Asp Pro Gln Ser Arg Asp Ala Arg Gln Gln Arg Phe Ala His Phe Thr                260                 265                 270    Glu Leu Ala Ile Ile Ser Val Gln Glu Ile Val Asp Phe Ala Lys Gln            275                 280                 285    Val Pro Gly Phe Leu Gln Leu Gly Arg Glu Asp Gln Ile Ala Leu Leu        290                 295                 300    Lys Ala Ser Thr Ile Glu Ile Met Leu Leu Glu Thr Ala Arg Arg Tyr    305                 310                 315                 320    Asn His Glu Thr Glu Cys Ile Thr Phe Leu Lys Asp Phe Thr Tyr Ser                    325                 330                 335    Lys Asp Asp Phe His Arg Ala Gly Leu Gln Val Glu Phe Ile Asn Pro                340                 345                 350    Ile Phe Glu Phe Ser Arg Ala Met Arg Arg Leu Gly Leu Asp Asp Ala            355                 360                 365    Glu Tyr Ala Leu Leu Ile Ala Ile Asn Ile Phe Ser Ala Asp Arg Pro        370                 375                 380    Asn Val Gln Glu Pro Gly Arg Val Glu Ala Leu Gln Gln Pro Tyr Val    385                 390                 395                 400    Glu Ala Leu Leu Ser Tyr Thr Arg Ile Lys Arg Pro Gln Asp Gln Leu                    405                 410                 415    Arg Phe Pro Arg Met Leu Met Lys Leu Val Ser Leu Arg Thr Leu Ser                420                 425                 430    Ser Val His Ser Glu Gln Val Phe Ala Leu Arg Leu Gln Asp Lys Lys            435                 440                 445    Leu Pro Pro Leu Leu Ser Glu Ile Trp Asp Val His Glu        450                 455                 460

or a degenerate variation thereof; the protein being free of other humanreceptor proteins.

A second embodiment concerns a DNA sequence encoding human steroidhormone receptor NER complementary DNA, said DNA sequence being free ofother human DNA sequences.

As will be appreciated by those of skill in the art, there is asubstantial amount of redundancy in the set of codons which translatespecific amino acids. Accordingly, the invention also includesalternative base sequences wherein a codon (or codons) are replaced withanother codon, such that the amino acid sequence translated by the DNAsequence remains unchanged. For purposes of this specification, asequence bearing one or more such replaced codons will be defined as adegenerate variation. Also included are mutations (exchange ofindividual amino acids) which one of skill in the art would expect tohave no effect on functionality, such as valine for leucine, argininefor lysine and asparagine for glutamine.

One class of the second embodiment of the invention concerns thefollowing nucleotide sequence (SEQ ID NO:1:) of complementary DNAdepicted from the 5' to the 3' terminus:

    CAAGAAGTGG CGAAGTTACC TTTGAGGGTA TTTGAGTAGC GGCGGTGTGT CAGGGGCTAA                                             60    AGAGGAGGAC GAAGAAAAGC AGAGCAAGGG AACCCAGGGC AACAGGAGTA GTTCACTCCG                                            120    CGAGAGGCCG TCCACGAGAC CCCCGCGCGC AGGCATGAGC CCCGCCCCCC ACGCATGAGC                                            180    CCCGCCCCCC GCTGTTGCTT GGAGAGGGGC GGGACCTGGA GAGAGGCTGC TCCGTGACCC                                            240    CACCATGTCC TCTCCTACCA CGAGTTCCCT GGATACCCCC CTGCCTGGAA ATGGCCCCCC                                            300    TCAGCCTGGC GCCCCTTCTT CTTCACCCAC TGTAAAGGAG GAGGGTCCGG AGCCGTGGCC                                            360    CGGGGGTCCG GACCCTGATG TCCCAGGCAC TGATGAGGCC AGCTCAGCCT GCAGCACAGA                                            420    CTGGGTCATC CCAGATCCCG AAGAGGAACC AGAGCGCAAG CGAAAGAAGG GCCCAGCCCC                                            480    GAAGATGCTG GGCCACGAGC TTTGCCGTGT CTGTGGGGAC AAGGCCTCCG GCTTCCACTA                                            540    CAACGTGCTC AGCTGCGAAG GCTGCAAGGG CTTCTTCCGG CGCAGTGTGG TCCGTGGTGG                                            600    GGCCAGGCGC TATGCCTGCC GGGGTGGCGG AACCTGCCAG ATGGACGCTT TCATGCGGCG                                            660    CAAGTGCCAG CAGTGCCGGC TGCGCAAGTG CAAGGAGGCA GGGATGAGGG AGCAGTGCGT                                            720    CCTTTCTGAA GAACAGATCC GGAAGAAGAA GATTCGGAAA CAGCAGCAGC AGGAGTCACA                                            780    GTCACAGTCG CAGTCACCTG TGGGGCCGCA GGGCAGCAGC AGCTCAGCCT CTGGGCCTGG                                            840    GGCTTCCCCT GGTGGATCTG AGGCAGGCAG CCAGGGCTCC GGGGAAGGCG AGGGTGTCCA                                            900    GCTAACAGCG GCTCAAGAAC TAATGATCCA GCAGTTGGTG GCGGCCCAAC TGCAGTGCAA                                            960    CAAACGCTCC TTCTCCGACC AGCCCAAAGT CACGCCCTGG CCCCTGGGCG CAGACCCCCA                                            1020    GTCCCGAGAT GCCCGCCAGC AACGCTTTGC CCACTTCACG GAGCTGGCCA TCATCTCAGT                                            1080    CCAGGAGATC GTGGACTTCG CTAAGCAAGT GCCTGGTTTC CTGCAGCTGG GCCGGGAGGA                                            1140    CCAGATCGCC CTCCTGAAGG CATCCACTAT CGAGATCATG CTGCTAGAGA CAGCCAGGCG                                            1200    CTACAACCAC GAGACAGAGT GTATCACCTT CTTGAAGGAC TTCACCTACA GCAAGGACGA                                            1260    CTTCCACCGT GCAGGCCTGC AGGTGGAGTT CATCAACCCC ATCTTCGAGT TCTCGCGGGC                                            1320    CATGCGGCGG CTGGGCCTGG ACGACGCTGA GTACGCCCTG CTCATCGCCA TCAACATCTT                                            1380    CTCGGCCGAC CGGCCCAACG TGCAGGAGCC GGGCCGCGTG GAGGCGTTGC AGCAGCCCTA                                            1440    CGTGGAGGCG CTGCTGTCCT ACACGCGCAT CAAGAGGCCG CAGGACCAGC TGCGCTTCCC                                            1500    GCGCATGCTC ATGAAGCTGG TGAGCCTGCG CACGCTGAGC TCTGTGCACT CGGAGCAGGT                                            1560    CTTCGCCTTG CGGCTCCAGG ACAAGAAGCT GCCGCCTCTG CTGTCGGAGA TCTGGGACGT                                            1620    CCACGAGTGA GGGCTGGGCC ACCCAGCCCC ACAGCCTTGC CTGACCACCC TCCAGCAGAT                                            1680    AGACGCCGGC ACCCCTTCCT CTTCCTAGGG TGGAAGGGGC CCTGGGCGAG CCTGTAGACC                                            1740    TATCGGCTCT CATCCCTTGG GATAAGCCCC AGTCCAGGTC CAGGAGGCTC CCTCCCTGCC                                            1800    CAGCGAGTCT TCCAGAAGGG GTGAAAGGGT TGCAGGTCCC GACCACTGAC CCTTCCCGGC                                            1860    TGCCCTCCCT CCCCAGCTTA CACCTCAAGC CCAGCACGCA GCGTACCTTG AACAGAGGGA                                            1920    GGGGAGGACC CATGGCTCTC CCCCCCTAGC CCGGGAGACC AGGGGCCTTC CTCTTCCTCT                                            1980    GCTTTTATTT AATAAAAATA AAAACAGAAA AAAAAAAAAA AAAAAAAAAA                                            2030

A third embodiment of this invention concerns systems for expressing allor part of the human steroid hormone receptor NER.

One class of this third embodiment of the invention comprises:

An expression construct, such as a plasmid which comprises:

a) an expression vector, such as PJ3NERI, and

b) a base sequence encoding human steroid hormone receptor NER protein.

Within this class of the third embodiment, the steroid hormone receptorNER comprises the nucleotide sequence (SEQ ID NO:1:) of complementaryDNA as shown above.

A second class of this third embodiment of the invention concerns asystem for the transient expression of human steroid hormone receptorNER in a suitable host cell, such as a monkey kidney cell line (COS),the system comprised of a vector which expresses human steroid hormonereceptor NER cDNA.

It is understood, and is readily apparent to those skilled in the art,that a wide variety of commonly used cell lines are suitable for use inthe present invention. Suitable cell lines derived from various speciesinclude, but are not limited to, cell lines of human, bovine, porcine,monkey, and rodent origin, or from yeast and bacterial strains.

A fourth embodiment of the invention concerns a method of using any ofthe above eukaryote or prokaryote expression systems for determining thebinding affinity of a test sample for steroid hormone receptor NER.

Following the isolation of a DNA sequence encoding human steroid hormonereceptor NER cDNA, a chimeric gene can be created by substituting theDNA-binding domain region in the DNA sequence encoding NER cDNA with aDNA-binding domain region taken from a DNA sequence coding for anothersteroid hormone receptor protein, e.g., glucocorticoid (GR) receptorprotein, thyroid receptor protein, mineral corticoid receptor protein orretinoic acid receptor protein. Next, a suitable receptor-deficient hostcell is transfected with: (1) the chimeric receptor gene, which ispreferably carried on an expression plasmid, and (2) a reporter gene,such as the CAT gene or the firefly luciferase gene, which is alsopreferably carried on a plasmid. In any case, the reporter gene isfunctionally linked to an operative hormone response element (HRE)(either wild-type or engineered) wherein the hormone response element iscapable of being activated by the DNA-binding domain used to make thechimeric receptor gene. (For example, if the chimeric receptor genecontains the DNA-binding domain region from glucocorticoid receptorcoding DNA, then the HRE should be a wild-type, an engineered, or asynthetic GRE, i.e., one that can be activated by the operative portionof the DNA-binding region of a GR receptor protein.) Next, thetransfected host cell is challenged with a test sample which containsone or more ligand(s) which can potentially bind with the ligand-bindingdomain region of the chimeric protein coded for by the chimeric gene. Todetermine the extent that ligands can functionally complex with thechimeric receptor protein, induction of the reporter gene is monitoredby monitoring changes in the protein levels of the protein coded for bythe reporter gene. (For example, if luciferase is the reporter gene, theproduction of luciferase is indicative of receptor-regulated genetranscription.) Finally, when a ligand(s) is found that can inducetranscription of the reporter gene, it is concluded that this ligand(s)can bind to the receptor protein coded for by the initial sample DNAsequence. This conclusion can be further verified by testing the bindingproperties of the receptor protein, coded for by the initial sample DNAsequences, vis-a-vis the ligand(s) that induce expression of thereporter gene.

The fourth embodiment further concerns a method for determining theaffinity of a test sample for activation of the steroid hormone receptorNER, the method comprising:

(a) constructing a chimeric gene by substituting portions of aDNA-binding domain region of a DNA sequence encoding human steroidhormone receptor NER cDNA with operative portions of a DNA-bindingdomain region from a known ligand-responsive receptor protein;

(b) introducing into a suitable receptor-deficient host cell:

(i) the chimeric gene from step (a), and

(ii) a reporter gene functionally linked to an operative hormoneresponse element wherein the hormone response element is capable ofbeing activated by the DNA-binding domain region of the receptor proteinencoded by the chimeric gene of step (a);

(c) challenging the transfected host cell from step (b) with the testsample to be evaluated for ligand-binding activity with the chimericreceptor protein encoded by the chimeric gene of step (a);

(d) assaying induction of the reporter gene by monitoring changes in theprotein levels of the protein coded for by the reporter gene.

One class of this embodiment concerns a method of using a monkey kidneycell line (COS) as the suitable receptor-deficient host cell. Inaddition, the COS host cell line may be transfected with a plasmid, theplasmid comprising:

(a) an expression vector, such as PJ3NERI, and

(b) the base sequence encoding human steroid hormone receptor NERprotein.

The aforementioned fourth embodiment is further useful for identifyingcompounds, such as TOFA, which compounds potentiate the effects ofligands for other receptors, such as the Dopamine D1 receptor and themuscarinic receptor. This embodiment is also useful in identifyingligands for new hormone systems which regulate bodily function.

Yet another class of this embodiment of the present invention comprisesligand dependent screening assays for assessing a compound or mixture ofcompounds to determine whether the compound (or any one of thecompounds) modulate the NER receptor. Ligand dependent screening assaysare performed by co-expressing the NER receptor and a reporter gene inwhich the transcription is under the control of the NER receptor. Such areporter gene can be the MMTV-luciferase reporter gene in which the MMTVpromoter is modified to be under the control of the NER receptor. Theplasmids containing cDNA for the NER steroid hormone receptor and theappropriate reporter gene can be transfected in to COS or other suitablecells. Ligands or extracts are added after the transfection and 18 to 48hours later, the cells are washed, cell extracts are prepared andassayed for luciferase activity. In some experiments, transfection isperformed by batch mode in large tissue culture dishes. After 18 hours,the cells are washed and seeded into multi-well plates. After cellsettling, the ligands are added and luciferase activities are tested oneor two days later. All compounds or extracts are dried and dissolved intheir appropriate solvent such as ethanol or DMSO as 100-1000 foldconcentrated stock solutions.

In overview, the present invention describes methods to isolate thehuman steroid hormone receptor NER complementary DNA (cDNA) withoutprior knowledge of its protein sequence or gene sequence. Polymerasechain reaction (PCR) technique was utilized for the isolation of humansteroid hormone receptor NER cDNA.

The complete sequence of the human steroid hormone receptor NER cDNA wasdetermined, and its encoded protein sequence was deduced. Among otherthings, such sequence information is useful in the process of developingnovel steroid hormone agonists and antagonists.

An expression system was used to express the cloned human steroidhormone receptor NER cDNA. The COS (a monkey kidney cell line)expression system can be used to measure the ligand binding propertiesof human steroid hormone receptor NER.

Assay protocols use the heterologously expressed human steroid hormonereceptor NER for determination of the activation of steroid hormonereceptor NER by antagonists.

The present invention generally relates to a new member of the steroidhormone receptor superfamily. The amino acid sequence deduced from theDNA sequence (Bases 245 to 1027) shows the characteristic features ofboth the DNA and the ligand binding domains of this family of receptors.Sequence analysis predicted a protein of 461 amino acids which includesthe conserved amino acid residues characteristic of the DNA andligand-binding domains of nuclear receptors.

This invention relates to NER, a new member of the steroid receptor-likegene family which was isolated from a human bone cell cDNA library. NERcodes for a polypeptide of 461 amino acids which contains the conservedsequences of the DNA and ligand binding domains of typical steroidreceptors. The best homology is shared with the different retinoic acidreceptors: α, β & γ, 55% at the DNA α, γ binding domain and 38-40% atthe ligand binding domain. A single transcript of 2.3 kb was detected inall cells and tissues tested. We tested the potential of theseconstructs to mediate ligand dependent transcription activation ofreporter genes.

The nuclear receptor-gene family is expanding in size, as new membersare constantly identified. Here we report the cloning of a new sequencefrom human osteosarcoma cells. This gene, named NER, codes for apolypeptide of 461 amino acids and contains the conserved sequencestypical of both the DNA and the ligand binding domains. The aminoterminus of the predicted protein contains a high number of proline andserine residues which might introduce a highly stabilized and complexsecondary structure. A high number of proline residues was also found inother nuclear receptor and other molecules with transcriptional activitysuch as CTf/N1, fos, jun. p53, OCT-2 and SRF (Mitchell & Tjian, Science,245, pp. 371-379 (1989); Mermod et al., 1989).

The size of the deduced protein and the spatial distribution of thedifferent domains resemble the arrangement found in the thyroid, vitaminD and retinoic acid-receptor subgroup (Lazar et al., Proc Natl. Acad.Sci. 86, pp. 7771-7774, 1989). The sequence homology at the predictedligand binding domain ranges between 33-40% identity with the members ofthis subgroup, while homologies lower than 25% were measured when theligand binding domain was compared to the corresponding domain of thesteroid receptor subgroup which includes the estrogen, glucocorticoid,androgen and progesterone. As mentioned above, the highest homology ofthe ligand binding domain was the retinoic acid receptors. Thishomology, 40% with retinoic acid receptor RARα is much lower thanhomologies of 79% and over which are found between RARα, RARβ, RARγ. Thedegree of sequence similarity, however, is not always indicative of thenature of the ligand as evident from the recently discovered new form ofretinoic acid receptor, RXR which shared only a 27% identity with theother retinoic acid receptors (Oro et al., Nature, 347, pp. 298-301,1990). It is thus impossible to assign or to exclude any of the knownligands based on sequence homology considerations. The homology at theDNA binding domain is around 50% with most other nuclear receptors. Thehighest degrees of homology were measured with estrogen and withretinoic acid receptors, 56%, and 53-55% respectively. However, theselevels were only marginally higher than the homologies with the otherreceptors. It is worth noting that the homology shared between thedifferent retinoic acid receptors (types α, β and γ) at this domain arehigher than 95%. And even the homology of RXR to the other retinoic acidreceptors at this region exceeds 60%.

Although cloned from an osteoblastic cell line, the mRNA for NER iswidely distributed in different tissues and in all the tested celllines.

To simplify the search for the elusive ligand, we constructed a hybridreceptor gene comprising the DNA binding domain of estrogen receptorlinked to the ligand binding domain of the NER gene. Such strategy wasproven successful in the identification of ligands for the PPARreceptor. Issenmann and Green, Nature, 347, pp. 645-649 (1990).

To search for putative ligands for the NER receptor, the chimericreceptor GR/NER was prepared. This chimeric receptor is capable ofexhibiting ligand dependent activation of transcription of aheterologous responsive DNA sequence. Chimeric receptors were preparedin which the amino terminal portion of the mouse glucocorticoid receptor(mGR) that includes the DNA binding domain was fused to the putativeligand binding domains of the nuclear receptor NER to form therespective chimeric receptor GR/NER. The cDNA sequences coding for aminoacid residues Arg¹⁵⁵ and Glu¹⁵⁶ of the hNER receptor (Shinar et al.,NER, a new member of a gene family encoding the steroid hormone nuclearreceptor; GENE (in press)) were converted to the Xho I restriction site,which site was later used for the ligation to the GR cDNA sequences thatcoded for the DNA binding domain. Thus, the amino acid residues Arg¹⁵⁵and Glu¹⁵⁶ of the NER receptor were converted to amino acid residuesSer¹⁵⁵ and His¹⁵⁶.

The chimera was employed in ligand dependent transactivation assays withthe reporter gene MMTV-luciferase, in which the luciferase cDNA istranscribed under the control of the MMTV promoter.

TOFA was identified as a ligand of the NER receptor via directedscreening of compounds topologically similar to fatty acids from theMerck Chemical Collection. The compounds were selected from the MerckChemical Structure Database by using Merck's topological similarity(TOPOSIM) program that is the result of unpublished work by SimonKearsley which is based on the similarity descriptors developed atLederle Laboratories (Raymond E. Charhart, Dennis H. Smith, R.Venkataraghavan, J. Chem. Inf. Comp. Science, 1985, 25:64-73) but whichincludes additional descriptors such as partial charge. About 250compounds were selected for testing in the transactivation assays. TOFAstimulated the expression mediated by the GR/NER, GR/NUC and GR/PPARhybrid receptors, in a dose dependent manner. In contrast to TOFA, theligands Wy14643 and oleic acid maintained their expected receptorspecificity, and activated expression mediated by GR/PPAR and GR/NUC butnot by the GR/NER receptors. However, TOFA did not act as a generalstimulator of transcription since it did not stimulate expression of theMMTV-luciferase reporter gene after co-transfection of the native NUCIand glucocorticoid receptors. The specific action of TOFA was furtherdemonstrated by the fact that TOFA did not stimulate the expression ofluciferase in cells transfected only with the MMTV-luciferase reportergene, or with a reporter gene in which the expression was under thecontrol of the early promoter of SV40.

These ligand dependent transcription assays and the binding assays tothe NER receptor may be used to identify additional compounds, includingthose that are more potent than TOFA or have better selectivity towardactivation of G protein-coupled receptors.

TOFA was found to activate transcription mediated by the ligand bindingdomain of the unrelated NUC and NER receptors. It is thus possible thatTOFA may be interacting indirectly with these receptors in a ligandindependent fashion akin to the interaction of dopamine with the COUP-TFreceptor (Power et al., Dopamine activation of an orphan of the steroidreceptor superfamily, Science 252(5012): 1546-1548, 1991; Power et al.,Dopaminergic and ligand-independent activation of steroid hormonereceptors, Science 254(5038): 1636-1639, 1991; Power et al., Newinsights into activation of the steroid hormone receptor superfamily,Trends Pharmacol. Sci. 13(8): 318-323, 1992). To gain information forthe interaction between TOFA and the D1 dopaminergic system, we testedthe in vitro and in vivo interaction between the dopamine receptoragonist dopamine, the dopamine D1 receptor antagonist SCH23390, and TOFAin vivo and in vitro. Contrary to the reports disclosed by Power et al.(supra) that the treatment of CV1 cells with dopamine activatestranscription mediated by steroid hormone receptors, treatment of CV1cells with dopamine did not stimulate the transcription mediated byGR/NER. Moveover, in COS cells that do not express the D1 dopaminereceptor, TOFA activated the transcription mediated by the GR/NERchimeric receptor. Furthermore, dopamine suppressed transcriptionmediated by GR/NER in CV1 cells but not in COS cells. Treatment withSCH23390 increased transcription and alleviated the suppression oftranscription induced by dopamine. These results indicate that ,although there is cross talk between TOFA and the dopaminergic system,TOFA activates the receptors via a different molecular mechanism thanthat suggested for the stimulation of steroid hormone receptors bydopamine. To gain information for the interaction between TOFA and theD₁ dopaminergic system in vivo, we tested the in vivo interactionbetween the dopamine receptor antagonist, SCH23390 and TOFA using thecatalepsy induced by the D₁ antagonists in rats. Pretreatment with TOFAmarkedly increased the SCH23390 catalepsy by at least 25 fold. Thecatalepsy induced by pilocarpine, an agonist of the muscariniccholinergic receptor was potentiated by about 3 fold by TOFApretreatment. However, the catalepsy induced by the D₂ dopamine receptorantagonist haloperidol was unaffected by TOFA pretreatment.

This invention also relates to a method of finding potentiators ofreceptors, particularly potentiators of dopamine D1 receptor antagonistsand of the muscarinic receptor. This method employs a screeningprocedure using the novel recombinant human steroid hormone receptor,NER.

The ligand screening assay used in the present method is describedbelow:

Ligand dependent transcription screening assays are performed byco-expressing the NER receptor and a reporter gene in which thetranscription is under the control of the NER receptor. Such a reportergene is preferably the MMTV-luciferase reporter gene in which the MMTVpromoter is modified to be under the control of the NER receptor. Theplasmids containing cDNA for the NER steroid hormone receptor and theappropriate reporter gene are transfected into COS or other suitablecells. Ligands or extracts are added after the transfection and 18 to 48hours later, the cells are washed, cell extracts are prepared andassayed for luciferase activity. Alternatively, transfection may beperformed by batch mode in large tissue culture dishes. After 18 hours,cells are washed and seeded into multi-well plates. After cell settling,the ligands are added and luciferase activities are tested one or twodays later. All compounds or extracts are dried and dissolved in theirappropriate solvent such as ethanol or DMSO as 100-1000-foldconcentrated stock solutions.

Further, this invention relates to the use of TOFA(5-(tetradecyloxy)-2-furan carboxylic acid), and pharmaceuticallyacceptable salts and esters thereof as a potentiator of inhibition ofthe dopamine D1 receptor and of the muscarinic receptor. TOFA was foundthrough the above screening procedure employing NER. TOFA activates NERand is a potentiator of ligands for other receptors. However, TOFA hasno independent effect on the receptors whose ligands are potentiated byTOFA.

TOFA appears to act through a novel mechanism of interaction with cellsurface receptor mechanisms via their intracellular signaling pathways.The use of the potentiator TOFA presents clear clinical advantages,since by circumventing direct effects at the neurotransmitter receptors,alteration to receptor up and down regulations which often compromisethe action of direct receptor-active drugs is avoided.

TOFA is particularly useful in the treatment of diseases for which thedopamine D1 antagonist SCH23390 is useful. For example, SCH23390prevents the addictive properties of cocaine and may be useful inprotecting or preventing the toxic effects which result from an overdoseof cocaine, amphetamines or other CNS stimulants. (Lomax, and Daniel,"Cocaine and body temperature in the rat: effect of dopamine D1antagonists", Proc. West Pharmacol. Soc. 34:5-9, 1991; and Witkin etal., "Interaction of haloperidol and SCH23390 with cocaine and dopaminereceptor subtype-selective agonists on schedule-controlled behavior ofsquirrel monkeys", Psychopharmacology Berl. 104(4):452-431, 1991.)Further, TOFA may also be useful in potentiating the effects of SCH23390in the treatment of schizophrenia and movement disorders including thosewhich develop during treatment with antipsychotic drugs. In addition,TOFA is useful in potentiating the effects of SCH23390 in preventing thedevelopment of intraocular pressure induced by dopamine agonists inhydrodynamic disorders of the eye (see, Virno et al., "Dopamine,dopaminergic drugs and ocular hypertension", Int. Ophthalmol.16(4-5):349-353, 1992), and in patients with an increase in intracranialpressure (see Boyson and Alexander, "Net Production of cerebrospinalfluid is decreased by SCH22390", Ann. Neurol. 27(6):631-635, 1990).

TOFA is also useful in potentiating the effects of SCH22390 in thetreatment of Alzheimer's disease.

Further, since TOFA markedly potentiates the differentiation effect ofNGF in PC12 cells and potentiates the in vivo effect of the cholinergicreceptor stimulant (pilocarpine), TOFA may also be beneficially employedin treating memory disorders associated with cholinergicdeficiency--both those associated with normal aging and in age-relateddiseases such as Alzheimer's disease.

Although TOFA is known to be a potent inhibitor of fatty acid synthesis,it is unlikely that this action is the shared mechanism through whichTOFA activates these receptors. Cerulenin, another compound which is apotent inhibitor of fatty acid synthesis (Kawaguchi et al., J. Biochem.Tokyo, 92:7-12 (1982)), does not mimic the potentiating effects of TOFA,adding further evidence to demonstrate that inhibition of fatty acidsynthesis per se is not responsible for the activation of the chimericreceptors and the cross talk with the dopamine receptor signalingpathway.

In summary, we have identified a new member of the steroid hormonereceptor superfamily. The identification of these functions may provideus with an insight into a novel hormonal regulated system.

The pharmaceutically acceptable salts of the compound of this inventioninclude those formed from cations such as sodium, potassium, aluminum,calcium, lithium, magnesium, zinc, and from bases such as ammonia,ethylenediamine, N-methyl-glutamine, lysine, arginine, ornithine,choline, N,N'-dibenzylethylenediamine, chloroprocaine, diethanolamine,procaine, N-benzylphenethylamine, diethylamine, piperazine,tris(hydroxymethyl)aminomethane, and tetramethylammonium hydroxide.These salts may be prepared by standard procedures, e.g., by reactingthe free acid with a suitable organic or inorganic base.

Esters of TOFA may be prepared by dissolving the TOFA in a dry organicsolvent, preferably tetrahydrofuran (THF) at 0-30° C. and treating withthe appropriately substituted isourea for 8-24 hours, cooling to -15° C.and filtering the urea. The filtrate is concentrated under reducedpressure to yield the desired ester. Especially suitable esters of thepresent invention include:

(a) C₁₋₅ alkyl, or

(b) C₁₋₅ alkyl substituted with

(i) phenyl, or

(ii) phenyl substituted with methyl, methoxy, Cl, Br, I, F or hydroxy;

however, other pharmaceutically acceptable esters may be employed.

Activators of NER, such as TOFA, can be administered in such oral dosageforms as tablets, capsules (each including timed release and sustainedrelease formulations), pills, powders, granules, elixirs, tinctures,suspensions, syrups and emulsions. Likewise, they may also beadministered in intravenous (both bolus and infusion), intraperitoneal,subcutaneous or intramuscular form, all using forms well known to thoseof ordinary skill in the pharmaceutical arts. An effective but non-toxicamount of the compound desired can be employed as an antiandrogenicagent.

The dosage regimen utilizing the activator of NER is selected inaccordance with a variety of factors including type, species, age,weight, sex and medical condition of the patient; the severity of thecondition to be treated; the route of administration; the renal andhepatic function of the patient; and the particular compound or saltthereof employed. An ordinarily skilled physician or veterinarian canreadily determine and prescribe the effective amount of the activator ofNER required to potentiate the effects of the dopamine D1 antagonist orthe muscarinic agonist, or to stimulate the production and effects ofNGF.

Oral dosages of the present invention, when used for the indicatedeffects, will range between about 0.01 to 100 mg/kg of the NERactivator, preferably 0.1 to 50 mg per day. The compositions arepreferably provided in the form of tablets containing 0.5, 1.0, 2.5,5.0, 10.0, 15.0, 25.0 and 50.0 mg of active ingredient. Advantageously,TOFA may be administered in a single daily dose, or even lessfrequently, or the total daily dosage may be administered in divideddoses of two, three or four times daily. Furthermore, TOFA can beadministered in intranasal form via topical use of suitable intranasalvehicles, or via transdermal routes, using those forms of transdermalskin patches well known to those of ordinary skill in that art. To beadministered in the form of a transdermal delivery system, the dosageadministration will, of course, be continuous rather than intermittentthroughout the dosage regimen. Other preferred topical preparationsinclude creams, ointments, lotions, aerosol sprays and gels, wherein theconcentration of active ingredient would range from 0.1% to 15%, w/w orw/v.

In the methods of the present invention, TOFA can form the activepotentiating ingredient, and is typically administered in admixture withsuitable pharmaceutical diluents, excipients or carriers (collectivelyreferred to herein as "carrier" materials) suitably selected withrespect to the intended form of administration, that is, oral tablets,capsules, elixirs, syrups and the like, and consistent with conventionalpharmaceutical practices.

For instance, for oral administration in the form of a tablet orcapsule, the active drug component can be combined with an oral,non-toxic pharmaceutically acceptable inert carrier such as ethanol,glycerol, water and the like. Moreover, when desired or necessary,suitable binders, lubricants, disintegrating agents and coloring agentscan also be incorporated into the mixture. Suitable binders includestarch, gelatin, natural sugars such as glucose or beta-lactose, cornsweeteners, natural and synthetic gums such as acacia, tragacanth orsodium alginate, carboxymethylcellulose, polyethylene glycol, waxes andthe like. Lubricants used in these dosage forms include sodium oleate,sodium stearate, magnesium stearate, sodium benzoate, sodium acetate,sodium chloride and the like. Disintegrators include, withoutlimitation, starch, methyl cellulose, agar, bentonite, xanthan gum andthe like.

Activators of NER such as TOFA can also be administered in the form ofliposome delivery systems, such as small unilamellar vesicles, largeunilamellar vesicles and multilamellar vesicles. Liposomes can be formedfrom a variety of phospholipids, containing cholesterol, stearylamine orphosphatidylcholines.

Activators of NER such as TOFA may also be delivered by the use ofmonoclonal antibodies as individual carriers to which the compoundmolecules are coupled. TOFA may also be coupled with soluble polymers astargetable drug carriers. Such polymers can includepolyvinylpyrrolidone, pyran copolymer,polyhydroxypropylmethacrylamidephenol,polyhydroxyethylaspartamidephenol, or polyethyleneoxidepolylysinesubstituted with palmitoyl residues. Furthermore, the TOFA may becoupled to a class of biodegradable polymers useful in achievingcontrolled release of a drug, for example, polylactic acid, polyepsiloncaprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals,polydihydropyrans, polycyanoacrylates and cross-linked or amphipathicblock copolymers of hydrogels.

Most preferably, activators of NER such as TOFA are administered incombination with compounds which themselves exhibit agonism orantagonism of G-coupled receptors, such as: pilocarpine, SCH23390,dihydroergocryptine, bromocryptine, metacholine, carbachol, betanechol,arecoline and oxotremorine, most preferably pilocarpine and SCH23390.

As used herein, the term "administration" refers to both concurrent andsequential administration of the NER activator and the potentiatedagents. Examples of such potentiated agents would include, but are notlimited to, dopamine D1 receptor antagonists and muscarinic receptoragonists. Illustrative of such dopamine D1 receptor antagonists are:SCH23390, dihydroergocryptine, and bromocryptine, and illustrative ofsuch muscarinic receptor agonists are: pilocarpine, metacholine,carbachol, betanechol, arecoline and oxotremorine. Dosages of dopamineD1 receptor antagonists range from 0.001 to 20 mg/kg, most preferably0.1 to 5 mg/kg. Dosages of muscarinic receptor agonists may beadministered topically for the treatment of elevated intraocularpressure in a 1 to 2% solution, or they may be administered orally orparenterally in dosage ranges from 0.001 to 20 mg/kg, most preferably0.1 to 5 mg/kg.

As used herein, "steroid hormone receptor superfamily" refers to theclass of related receptors comprised of glucocorticoid,mineralocorticoid, progesterone, estrogen, estrogen-related, vitamin D₃,thyroid, v-erb-A, retinoic acid and E75 (Drosophilia) receptors. As usedherein "steroid hormone receptor" refers to members within the steroidhormone receptor superfamily.

As used herein, "ligand" means an inducer, such as a hormone or growthsubstance. Inside a cell, the ligand binds to a receptor protein,thereby creating a ligand-receptor complex, which in turn can bind to anappropriate hormone response element. Single ligands may have multiplereceptors.

As used herein, the term "potentiator" means an agent or mechanism whichenhances the actions of a second agent or mechanism. The term"potentiating amount" refers to the amount of potentiatior that must beadministered in order to produce the potentiating effect in a subject.

The term "pharmacologically effective amount" shall mean that amount ofa drug or pharmaceutical agent that will elicit the biological ormedical response of a tissue, system, animal or human that is beingsought by a researcher or clinician.

As used herein, "expression construct" refers to a plasmid or vectorcomprising a transcriptional unit comprising an assembly of (1) agenetic element or elements having a regulatory role in gene expression,for example, promoters or enhancers, (2) a structural or coding sequencewhich is transcribed into mRNA and translated into protein, and (3)appropriate transcription initiation and termination sequences."Recombinant expression system" means a combination of an expressionconstruct and a suitable host microorganism. As used herein, the term"receptor" refers to a binding or recognition site with a specificmolecular configuration on a cell surface or within a cell structure,which causes a physiologic response upon stimulation by aneurotransmitter or other chemical, including a drug or toxin.

As used herein, the term "G-coupled receptor" refers to a receptor thatwhen activated is coupled to a membrane protein which binds GTP(G-protein) on its cytoplasmic surface and couples the activatedreceptor with adenylate cyclase. "Adenyl cyclase coupled receptor"refers to a receptor that when activated directly activates the enzymeadenylate cyclase.

The following examples are given for the purpose of illustrating thepresent invention and shall not be construed as being limitations on thescope or spirit of the instant invention.

EXAMPLE 1

Primers design

Degenerate DNA primers were designed to recognize the consensussequences of the DNA and ligand binding domains of a typical nuclearreceptor. The 5' primer ES11, (Seq. ID. No. 3) was degenerate oligomer5' TGTGAGGGCTGCAA(G/A)G(C/G)C, based on the conserved amino acidsCEGCKA(G) of the DNA binding domain. A second 5' primer, ES12, (Seq. ID.No. 4) TGTGAGGGCTGCAA(G/A)G(C/G)CTTCTTC contains six additionalnucleotides at its 3'-end corresponding to two conserved phenylalanineresidues following the CEGCKA(G) sequence. The antisense primer, ES15(Seq. ID. No. 5) AA(G)A(C,T,G)CCA(C,T,G)GGIAIIIIC(T)TTT(A,G,C)GC(G)TT,was designed to complement the semiconserved amino acid sequenceFAKxxPGF of the ligand binding domain of a typical receptor. Thenucleotides corresponding to the nonconserved amino acids (xx) weresubstituted with inosine (I) residues.

PCR Amplification

To use the polymerase chain reaction (PCR) method, degenerateoligonucleotides were synthesized according to the amino acid sequenceof two conserved segments shared by members of the nuclear receptorsuperfamily (R M Evans, Science 240:899-895 (1988)). The 5' end primers,ES11 and ES 12, were designed according to a segment of the DNA bindingdomain. The primer at the 3' end, ES 15, was prepared according to aconserved amino acid sequence in the ligand binding domain of theretinoid receptor subfamily and the vitamin D receptor. Since thisconserved region contains two nonconserved amino acid residues, inosinenucleotides were used as part of this primer. Human cDNA prepared frommRNA of osteosarcoma cells SAOS-2/B10, amplified with the primers ES11and ES15, yielded multiple DNA fragments with various sizes after thefirst round of amplification. A portion of the reaction was subjected toa second round of amplification using the nested primer ES12 and thesame 3' end primer ES15.

A random primed cDNA library was prepared from 2 μg total RNA isolatedfrom the osteosarcoma SAOS-2/B10 cells by the Moloney reversetranscriptase enzyme RTH according to the manufacturer recommendations(Bethesda Research Laboratories). The cDNA reaction (25 μL) was dilutedinto 300 mL water and heat denatured at 95° C. for 5 minutes and quicklychilled on ice. The cDNA (2.5 μL) and the first primer pair, ES11 andES15 (0.5 μM each) were employed in the amplification reaction with theamplitaq kit and the DNA thermal cycler (Perkin-Elmer-Cetus).

Primer ES11 has the following sequence (SEQ ID NO:3:): CGAATTCTGTGAGGGCTGCA ARGSC 25

wherein:

R represents A or G; and

S represents C or G;

and Primer ES15 has the following sequence (SEQ ID NO:5:): GGAATTCRAANCCNGGNANN NNYTTNGCRA A 31

wherein:

N (at the 11, 14 & 26 positions) represents A or C or G or T; N (at the17, 19, 20, 21 & 22 positions) represent inosine;

R represents A or G; S represents C or G; and Y represents C or T.

The following amplification cycles were conducted: denaturation at 94°C., 1.5 minutes; annealing at 65° C., 3 minutes; extension at 72° C., 5minutes for 3 cycles; denaturing at 94° C., 1 minute; annealing at 60°C., 3 minutes; extension at 72° C., 5 minutes for 15 cycles; anddenaturing at 94° C., 1 minute; annealing at 57° C., 3 minutes;extension at 72° C., 5 minutes for 20 cycles.

After completion of the first round of amplification, 5 μL of thereaction was added to an amplification reaction buffer containing asecond set of primers: a partially nested oligomer ES12 and the same 3'end primer ES15 (0.5 μM each).

Primer ES12 has the following sequence (SEQ ID NO:4:): CGAATTCTGTGAGGGCTGCA ARGSCTTCTT C 31

wherein:

R represents A or G; and

S represents C or G.

The second round of amplification was performed with the same programused for the first amplification cycles. The amplification products wereseparated on 5% polyacrylamide gel and stained by ethidium bromide. TheDNA products were isolated from the gel, phosphorylated by T4polynucleotide kinase and cloned into PUC 18 vector by blunt endligation. Clones were identified by digestion of plasmid DNA with PvuIIenzyme. The DNA insert was analyzed by double-stranded DNA sequencing bythe dideoxy termination method using sequenase enzyme kit (United StatesBiochemicals).

This amplification produced two major DNA fragments of 270 bp and 320,respectively.

EXAMPLE 2

Cloning and Sequencing of cDNA

The fragments from PCR amplification were cloned into plasmids andsequenced. The amino acid residues predicted by the DNA sequences,indicated that both DNA fragments may code for genuine and novelreceptors belonging to the steroid hormone superfamily. To obtain thecomplete cDNA clone the amplified cDNA fragment of 270 bp NER was usedfor the screening of a human osteosarcoma SAOS-2/B10 cells cDNA library.All the highly positive clones were identical and matched the sequencefor the amplified NER DNA fragment.

A human oligo-dT cDNA library was constructed RNA isolated fromosteosarcoma SAOS-2/B10 cells using the Lambda Librarian cloning kit(Invitrogen Corp.). Several positive clones were identified by plaquescreening with the ³² P! labeled DNA probe of the cloned amplifiedproduct (NER). The hybridization conditions were as described by ASchmidt, et al., J Biol Chem 259:7411-7415 (1984). The cDNA inserts werecloned into EcoRI site of the cloning vector PUC18. The complete DNAsequence of both strands was determined by the dideoxy sequencing methodusing a series of oligonucleotides synthesized as the DNA sequence databecame available.

Cloning of NER

The amplification of the cDNA prepared from the RNA of Saos-2/B10osteoblastic cell line with the ES11 and ES15 primers yielded multiplefragments after 40 rounds of amplification. Five percent of the firstamplification reaction were subjected to additional 30 rounds ofamplication with ES12 and ES15 oligomers. Primer ES12 that replaces ES11is six nucleotides longer and codes for two conserved phenylalanineresidues at the 3'-end, thus introduces an additional level ofspecificity to the amplification reaction. The second amplification stepresulted in the elimination of all but two DNA fragments. The twofragments; nuc-I, 320 bp, and Ner, 270 bp, were subcloned and sequenced.Sequence analysis revealed that both DNA fragments resemble the typicalDNA binding domain of steroid hormone receptor genes, but were notidentical to any of the known sequences.

Surprisingly, none of the two fragments contained sequences of theligand binding domain as could be predicted by the use of the ES15primer derived from that region. It was later realized that the 5' ES12oligomer primed the reaction at both directions although it shared only53% homology with that sequence.

In order to obtain full length cDNA clone for the novel putative nuclearreceptor NER, we screened a cDNA library from the Saos-2/B10 cells withthe NER amplified DNA fragment. Several clones were identified andcloned into pUC18 vectors. One of the largest clones, nuc-2-103 of 2 kbwas thoroughly analyzed and the nucleotide sequence and the predictedamino acid sequence were determined.

Sequencing of the NER clone revealed a long open reading frame codingfor a polypeptide of 461 amino acids. The deduced protein resembles inits structure a typical steroid-like receptor. At position 87-154, aputative "double zinc finger" structure which can serve as the DNAbinding domain was identified. Amino acid sequences that characterizethe ligand binding domain were located toward the carboxy terminus ofthe protein and were spaced like in the thyroid or retinoic acidreceptors. Comparing the sequence of the deduced protein with otherknown receptor sequences revealed that the DNA binding domain shared50-56% homology with all the steroid-like receptors. Highest scores atthis domain were: 56% for the estrogen receptor, 55% for the retinoicacid gamma receptor and mineralcorticoid receptor and 54% for retinoicacid A and glucocorticoid receptors. The ligand binding domain which isless conserved showed highest homology levels of 38-40% with the 3 typesof retinoic acid receptors, RARα, RARβ and RARγ 38% with vitamin Dreceptor and 33% with thyroid hormone receptor. The homology to theligand binding domains of estrogen, androgen, glucocorticoid andmineralocorticoid at this domain was significantly lower. The RXRretinoic acid receptor type X showed an intermediate value of 28%homology at this domain.

It is noteworthy that the amino acid terminus of NER (amino acids 1-87)contains high number of 17 proline residues and 10 serines.

EXAMPLE 3

Northern Blot Analysis

RNA from various tissues or the listed cell lines were prepared by usingguanidine thiocyanate or by the guanidine hydrochloride method (GGANemeth, et al., Anal Biochem 183:301-304 (1989); J M Chirgwin, et al.,Biochemistry, 18:5294-5299 (1979)). RNA samples were analyzed byformaldehyde agarose gel electrophoresis as described by (K M Rosen, etal., Focus 12:23-24 (1990)). The RNA was transferred by blotting toN-Hybond (Amersham Corp.), and hybridized with ³² P-labeled cDNA of NERas described by (A Schmidt, et al., J Biol Chem 259:7411-7415 (1984); KM Rosen, et al., Focus 12:23-24 (1990)).

Total RNA was extracted from rat or baboon tissues and processed forelectrophoresis and blot hybridization with ³² p labeled probe of Ner-Iby conventional methods as described by Fritsch et al., (1989).

Analysis of RNA from the osteoblastic Saos-2/B10 cells with the NERlabeled DNA probe revealed a single transcript of approximate 2.3 kb.Similar RNA transcripts were detected in all cell lines tested. Noapparent variations in size of the mRNA molecules could be observedbetween RNAs isolated from different species. Tissue distribution of theNER gene expression was examined by Northern hybridization. NER RNAtranscripts were detected in all the rat tissues or cells which weretested. Similar results were obtained with RNA isolated from tissues ofadult baboons.

Screening the Saos-2/B10 cDNA library with the labeled amplified DNAfragment encoding part of the putative novel nuclear receptor NERresulted in several positive cDNA clones. Sequence analysis of thepositive clones revealed that in addition to the expected full lengthcDNA clone for NER receptor we obtained two clones in which the DNAsequences differed from the expected NER putative receptor. The sequenceof one clone, named pE1001, matched the sequence of the known retinoicacid receptor type alpha (RARα), (Giguere et al., Nature 331, pp. 91-94,1987). Sequence analysis of the second clone (pE1005), revealed thecharacteristics of a novel nuclear receptor published and characterizedas a novel retinoic acid receptor X, (RXRα) (Mangelsdorf et al., Nature345, pp. 224-229 1990). Thus, these results illustrate that the cDNA forNER receptor can be utilized as an assay tool to identify known andnovel members of the class of steroid hormone nuclear receptors.

EXAMPLE 4

Activation of GR/NER, GR/NUC and GR/PPAR hybrid receptors by TOFA

To identify the putative ligand for NER receptor, the potential of NERto induce transcription of a reporter gene which contains induciblehormone responsive elements was examined. Several responsive elementswere tested; the thyroid/retinoic acid, estrogen, vitamin D and theglucocorticoid/progesterone elements. Transfection experiments in CV-1and L cells, revealed no ligand-dependent induction of the CAT reportergene. To facilitate the search for a ligand, hybrid receptor moleculeswere constructed.

The ligand dependent transcription assays were as described below:

Similar hybrid receptors GR/NER, GR/NUC and GR/PPAR, in identicalexpression plasmid backgrounds were prepared essentially as describedfor the construction of the GR/mPPAR and GR/NUC chimeric receptors inSchmidt et al., "Identification of a new member of the steroid hormonereceptor superfamily that is activated by a peroxisome proliferator andfatty acids," Mol. Endocrinol., 6(10):1634-41, 1992, and in Boie et al.,"Enantioselective activation of the peroxisome proliferator-activatedreceptor", J. Biol. Chem. 269(8):5530-4, 1993.

Briefly, the amino terminal portion of the mouse glucocorticoid receptor(mGR), that includes the DNA binding domain was fused to the putativeligand binding domains of the nuclear receptor NER, hNUC-I (Schmidt etal., 1992), mPPAR (Issenmann and Green, 1990) to form the respectivechimeric receptors GR/NER, GR/NUC and GR/PPAR. The GR/NER chimericreceptor was constructed as described for the GR/NUC and GR/PPAR hybridreceptors (Schmidt et al., 1992; Boie et al). The cDNA sequences codingfor amino acids Arg¹⁵⁵ and Glu¹⁵⁶ of the NER receptor were converted tothe Xho I restriction site, that was later used for the ligation to theGR cDNA sequences that coded for the DNA binding domain. Thus,converting amino acid residues Arg¹⁵⁵ and Glu¹⁵⁶ of NER receptor toamino acid residues Ser¹⁵⁵ and His¹⁵⁶.

The cDNA of the human NUCI receptor (pJ3NUCI) and the native mouseglucocorticoid receptor (pSV2WREC) were expressed under the control ofSV40 based expression vectors (Schmidt et al., 1992; Boie et al., 1993).The reporter gene was the plasmid pJA358 in which the expression offirefly luciferase is regulated by two tandem repeats of theglucocorticoid hormone response element (GRE) linked to the MMTVpromoter (Boie et al., 1993).

Transient transfection of COS and CV1 cells was performed as described(Schmidt 1992). Cells were plated (1.5×10⁵ in 1 mL) into 12 well dishesin phenol red-free medium supplemented with activated charcoal treatedfetal calf serum. The next day 0.12 mL of DNA 10 μg/ml (5 μg receptorDNA and 5 μg reporter plasmid), as a calcium phosphate precipitate, wasadded to the cells. Ligands were added to the cells 30 minutes aftertransfection. The next day (18 hours), the cells were washed and freshligands added. Twenty-four hours later, cell extracts were prepared andassayed for luciferase enzyme activity using the luciferase assay system(Promega Madison, Wis.). Each transfection was performed in triplicateand the luciferase activity of each sample measured in duplicate usingthe AutoClinilumat (Berthold Nashua, New Hampshire).

The transfected cells were treated with TOFA at 2 μM, 10 μM, 20 μM, and50 μM and oleic acid at 50 μM, and 300 μM. The results are depicted inTable 1, below.

                                      TABLE 1    __________________________________________________________________________    Activation of GR/NUC, GR/PPAR, GR/NER, NUC-I and GR Receptors by TOFA            FOLD OF STIMULATION    Treatment         μM            GR/NER                  GR/NUC                        GR/PPAR                               NUC-I GR    __________________________________________________________________________    Control         0  1.00 ± 0.13                  1.00 ± 0.09                        1.00 ± 0.19                               1.00 ± 0.40                                     1.00 ± 0.17    TOFA 2  2.35 ± 0.09                  1.95 ± 0.14                        2.89 ± 0.13                               0.83 ± 0.12                                     1.02 ± 0.40    TOFA 10 3.66 ± 0.40                  2.85 ± 0.20                        4.08 ± 0.10                               0.94 ± 0.08                                     0.80 ± 0.14    TOFA 20 4.82 ± 0.28                  ND    5.75 ± 0.14                               ND    ND    TOFA 50 ND    3.19 ± 0.20                        ND     1.27 ± 0.06                                     0.91 ± 0.30    Oleic Acid         50 0.90 ± 0.13                  ND    2.87 ± 0.2                               ND    ND    Oleic Acid         150            ND    3.42 ± 0.28                        ND     0.55 ± 0.08                                     0.44 ± 0.17    Oleic Acid         300            0.73 ± 0.21                  ND    14.69 ± 0.06    WY14643         100            0.57 ± 0.37                  ND    6.19 ± 0.44    __________________________________________________________________________

TOFA stimulated expression mediated by GR/NUC, GR/PPAR and GR/NER hybridreceptors in a dose-dependent manner. In contrast to TOFA, the ligandsWy14643 and oleic acid maintained their expected receptor specificityand activated expression mediated by GR/PPAR and GR/NUC, but not by theGR/NER chimera. However, TOFA did not act as general stimulator oftranscription since it did not stimulate expression of theMMTV-luciferase reporter gene after co-transfection of the native NUCIthat does not interact with the MMTV-luciferase reporter gene, andglucocorticoid receptors. The specific action of TOFA was furtherdemonstrated by the fact that TOFA did not stimulate the expression ofluciferase in cells transfected only with the MMTV-luciferase reportergene, or with a reporter gene in which the expression was under thecontrol of the promoter of SV40.

EXAMPLE 5

Activation of GR/PPAR by fatty acid inhibitors

The ligand transcription assay mediated by GR/PPAR in COS cells wasperformed as described in Example 4. The cells were treated with TOFAand Cerulenin at 0.1, 1, 10 and 100 μM. Concentrations of ceruleninabove 10 μM were toxic to the cells.

TOFA was originally developed as an inhibitor of fatty acid synthesis(Parker et al., 1977). We therefore tested whether cerulenin, aninhibitor of fatty acid synthesis which is structurally unrelated toTOFA, can mimic the transcriptional activation profile which was inducedby TOFA. At concentrations that inhibit fatty acid synthesis, cerulenindid not stimulate transcription mediated by GR/PPAR. These resultssuggest that the inhibition of fatty acid synthesis per se is unlikelyto be responsible for the action of TOFA on this receptor family.

The results are depicted in Table 2, below.

                  TABLE 2    ______________________________________    Activation of GR/PPAR Chimeric Receptor by Fatty Acid Inhibitors                 Fold of Stimulation    Treatment (μM)                   TOFA      Cerulenin    ______________________________________    0               1.0 ± 0.14                              1.0 ± 0.14    0.1            1.04 ± 0.14                             1.03 ± 0.12    1.0            2.38 ± 0.16                             1.13 ± 0.05    10.0           4.43 ± 0.23                             0.58 ± 0.11    100            4.10 ± 0.52                             --    ______________________________________

EXAMPLE 6

Activation of GR/NER by dopamine and TOFA in CV1 cells

TOFA was found to activate transcription mediated by the ligand bindingdomain of the members of the PPAR family (NUC-I and PPAR) and theunrelated NER receptor. It is thus possible that TOFA may be interactingindirectly with these receptors in a ligand independent fashion akin tothe interaction of dopamine with the COUP-TF and progesterone receptors(Power et al., 1991, 1991, 1992) that results with stimulation oftranscription mediated by these receptors in CV1 cells. We thereforetested the activation of GR/NER by TOFA and dopamine in CV1 cells thatexpress functional dopamine D1 receptors as measured by the elevation ofcAMP levels win response to treatment with dopamine. TOFA stimulated theexpression of luciferase mediated by the GR/NER chimera in CV1 cells.

The ligand dependent transcription assays were performed as described inExample 4, using CV1 cells in place of COS cells. The transfected CV1cells were treated with dopamine, TOFA, or with a combination of TOFAand dopamine at the indicated concentrations and tested for luciferaseactivity.

The results are depicted in Table 3 below. The results indicate thatdopamine did not stimulate transcription mediated by GR/NER as expectedfrom its effects on COUP-TF and progesterone receptors. In contrast, wefind that dopamine suppressed the transcription mediated by the GR/NERchimeric receptor. Furthermore, it partially inhibited the stimulationof transcription by TOFA. This suppression was not seen when GR/NER wastransfected into COS-7 cells that do not express significant levels ofthe dopamine D1 receptors. These results indicate that there is crosstalk between the dopamine receptor signally pathway and the NER receptorpathway.

                  TABLE 3    ______________________________________    Activation of GR/NER in CV1 Cells    Treatment (μM)    Dopamine   TOFA        Fold of Stimulation    ______________________________________    0          0           1.00 ± 0.08    0.1        0           1.28 ± 0.05    1.0        0           0.88 ± 0.11    10         0           0.55 ± 0.06    100        0           0.44 ± 0.14    100        10          1.40 ± 0.05    100        100         1.60 ± 0.07    0          100         3.70 ± 0.06    ______________________________________

EXAMPLE 7

Activation of GR/NER by dopamine and TOFA in COS-7 cells

The ligand dependent transcription assays were performed as described inExample 4. The transfected COS cells were treated with dopamine, TOFA,or with the combination of TOFA and dopamine at the indicatedconcentrations and tested for luciferase activity.

The results are depicted in Table 4 below.

                  TABLE 4    ______________________________________    Activation of GR/NER in COS-7 Cells    Treatment (μM)    Dopamine   TOFA        Fold of Stimulation    ______________________________________    0          0           1.00 ± 0.07    0.1        0           1.10 ± 0.07    1.0        0           0.87 ± 0.06    10         0           0.84 ± 0.08    100        0           0.87 ± 0.11    100        10          3.24 ± 0.06    100        100         2.85 ± 0.07    0          100         3.36 ± 0.04    ______________________________________

EXAMPLE 8

Effect of SCH 23390, a Dopamine D1 receptor antagonist, on theactivation of luciferase expression in GR/NER

To further characterize the suppression of luciferase expression bydopamine, we tested whether a D1 dopamine receptor antagonist,SCH-23390, can prevent the suppression induced by dopamine. CV1 cellswere co-transfected with the MMTV-Luciferase reporter gene (pJA358) andthe GR/NER chimeric receptor as described in Example 4. The cells weretreated with increasing amount of the dopamine D1 receptor antagonistSCH 23390 with or without 50 μM dopamine.

The results depicted in Table 5 below indicate that treatment withSCH-23390 can reverse the inhibition of luciferase expression caused bydopamine. Moreover, the D1 dopamine antagonist SCH-23390 stimulated thetranscription mediated by GR/NER. This further confirms the potentialinteraction between the NER receptor and dopamine receptor signalingpathways.

                  TABLE 5    ______________________________________    Effect of SCH-23390 on the transcription mediated by the chimeric    GR/NER receptor    Treatment (μM)                  Fold of Stimulation    SCH-23390     Control   Dopamine (50 μM)    ______________________________________    0.1           1.32 ± 0.15                            0.59 ± 0.06    1.0           1.22 ± 0.11                            0.55 ± 0.09    10.0          1.64 ± 0.38                            0.83 ± 0.12    100           2.38 ± 0.19                            2.61 ± 0.86    ______________________________________

EXAMPLE 9

Stimulation of neurite differentiation of PC12 cells by TOFA

To further study the influence of TOFA on the nervous system, we testedthe effect of TOFA on the differentiation of PC12 cells. PC12 cells wereplated at a density of approximately 100-200/mm² on collagen coatedplates or dishes in RPMI medium supplemented with 10% horse serum, 5%fetal calf serum, 50 g/mL streptomycin, 50 U/mL penicillin in a watersaturated atmosphere of 95% air and 5% CO₂, at 37° C. overnight. Mediumwas replaced with fresh RPMI containing 1.5% serum, 100 ng/mL NGF andthe indicated concentration of TOFA or an equivalent volume of vehicle(DMSO) giving a final concentration of 0.1% DMSO.

At different time points during the treatment, cells were photographedor fixed with 10% formalin. Morphology was examined after four days oftreatment with TOFA or vehicle (DMSO). The number of cells bearingneurites and average length of neurites were determined with the aid ofthe Bioquant imaging system. Treatment with TOFA alone did not induceneurite outgrowth in PC12 cells (data not shown). However, as depictedin Table 6 below, NGF alone induced a 15% increase in the number ofcells bearing neurites and these outgrowths reached a mean length of 20μm . Treatment with NGF and TOFA increased the percentage of cellsbearing neurites to 78%. Maximal neurite generation is observed at aconcentration of 10 μM TOFA. The addition of TOFA also increased thelength of the developed neurites in a dose related fashion.

                  TABLE 6    ______________________________________    Effects of TOFA on the Differentiation of PC-12 Cells                % Cells Bearing                         Average length of                Neurites neurites (μm)    ______________________________________    Vehicle       14.98 ± 1.68                             19.78 ± 2.01    0.1 μM TOFA                  14.27 ± 3.51                             23.67 ± 2.86    1.0 μM TOFA                  24.07 ± 3.06                             28.63 ± 3.92    10 μM TOFA .sup. 78.39 ± 4.89.sup.a                             .sup. 32.43 ± 2.38.sup.a    ______________________________________

Cells were treated with vehicle (0.1% DMSO) or various concentrations ofTOFA in the presence of 100 ng/mL NGF for 4 days, and then fixed with10% formalin. At least eighty cells from eight wells were analyzed ineach group. The results are shown as the mean±SEM. ^(a) p<0.01, versusvehicle by Dunnetts test.

EXAMPLE 10

Effect of TOFA on a Dopamine D1 Receptor antagonist (SCH 23390) InducedCatalepsy

Rats were pretreated with 0.4 mg/kg TOFA or with carrier (S.C.).Twenty-four hours later, the rats were challenged with 0.5 mg/kgSCH23390, a Dopamine D1 antagonist, and the duration of catalepsy wasmonitored.

Catalepsy was determined by a modification of the Bar method (Undie andFriedman, 1988). A steel bar, 1.1 cm in diameter and 50 cm long, wassuspended at a height of 10 cm above the working surface. Three sides ofthe bench around the bar were walled off with brown cardboard, and thelinings on the bench surface were selected to be of the same color withthe cardboard walls. The animal's hind limbs were freely placed on thebench, the tail laid out to the back, and the forelimbs placed over thebar. The length of time a rat stayed up on the bar was noted up to apreset cut-off point of 120 s. The time was counted off upon observingany of the following actions: (1) the animal came off, placing its twoforepaws on the bench, (2) the animal climbed onto or over the bar withboth hind limbs, (3) the animal initiated locomotion along or around thebar. Subsequently, time readings were translated into scores by awardinga score of 1 for each successfully completed 5 s on the bar. For eachobservation time, catalepsy scores were averaged for all the animals ina group to give the Mean Score of the group at that observation time.Further, the scores for each animal at all the observation times wereadded to give the total score for that animal. The mean of this lastgroup of numbers was called the Mean Total Score of the group.

Catalepsy scores were analyzed by computer in accordance with thestatistical procedures outlined by Winer, B. J.: Statistical principlesin experimental design. McGraw-Hill, New York, pp. 10-429, 1971.Generally, for a given set of comparisons, observations were subjectedto an appropriate analysis of variance followed by two-tailed Dunnett orStudent's t-tests to detect differences between treatment groups.

The data are shown in Table 7 below:

                  TABLE 7    ______________________________________    Effect of TOFA on SCH 23390 Induced Catalepsy    Pretreatment           Catalepsy    (Time = 0)   Treatment Duration (seconds)    ______________________________________    TOFA         none      0    control      SCH 23390 3.1 ± 1.3    TOFA         SCH 23390   75 ± 17.8    ______________________________________

The data shown above indicate that TOFA pretreatment markedly increasedthe duration of the SCH 23390-induced catalepsy by at least 25 fold.

EXAMPLE 11

Effect of TOFA on a Muscarinic Receptor Agonist (Pilocarpine) InducedCatalepsy

Rats were pretreated with 0.4 mg/kg TOFA or with carrier (S.C.).Twenty-four hours later, the rats were challenged with 16 mg/kgPilocarpine, an agonist of the muscarinic cholinergic receptor, and theduration of catalepsy was monitored as described in Example 10.

The data are shown in Table 8 below:

                  TABLE 8    ______________________________________    Effect of TOFA on Pilocarpine Induced Catalepsy                           Catalepsy    Pretreatment Treatment Duration (seconds)    ______________________________________    TOFA         none      0    control      Pilocarpine                           27.3 ± 3.6    TOFA         Pilocarpine                           84.1 ± 9.6    ______________________________________

The data shown above indicate that TOFA pretreatment increased theduration of the Pilocarpine-induced catalepsy by 3 fold.

EXAMPLE 12

Effect of TOFA on a Dopamine D2 Receptor Antagonist (Haloperidol) DrugInduced Catalepsy

Rats were pretreated with 0.4 mg/kg TOFA or with carrier (S.C.).Twenty-four hours later, the rats were challenged with 0.1 mg/kgHaloperidol, an antagonist of the Dopamine D2 Receptor, and the 30duration of catalepsy was monitored as described in Example 10.

The data are shown in Table 9 below:

                  TABLE 9    ______________________________________    Effect of TOFA on Haloperidol Induced Catalepsy                           Catalepsy    Pretreatment Treatment Duration (seconds)    ______________________________________    TOFA         none      0    control      Haloperidol                           20.3 ± 6.7    TOFA         Haloperidol                           28.9 ± 4.5    ______________________________________

The data shown above indicate that TOFA pretreatment did notsignificantly affect Haloperidol-induced catalepsy.

While the foregoing specification teaches the principles of the presentinvention, with examples provided for the purpose of illustration, itwill be understood that the practice of the invention encompasses all ofthe casual variations, adaptations, modifications, deletions, oradditions of procedures and protocols described herein, as come withinthe scope of the following claims and its equivalents.

    __________________________________________________________________________    #             SEQUENCE LISTING    - (1) GENERAL INFORMATION:    -    (iii) NUMBER OF SEQUENCES: 5    - (2) INFORMATION FOR SEQ ID NO:1:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 2030 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA (genomic)    -    (iii) HYPOTHETICAL: NO    -     (iv) ANTI-SENSE: NO    #ID NO:1: (xi) SEQUENCE DESCRIPTION: SEQ    - CAAGAAGTGG CGAAGTTACC TTTGAGGGTA TTTGAGTAGC GGCGGTGTGT CA - #GGGGCTAA      60    - AGAGGAGGAC GAAGAAAAGC AGAGCAAGGG AACCCAGGGC AACAGGAGTA GT - #TCACTCCG     120    - CGAGAGGCCG TCCACGAGAC CCCCGCGCGC AGGCATGAGC CCCGCCCCCC AC - #GCATGAGC     180    - CCCGCCCCCC GCTGTTGCTT GGAGAGGGGC GGGACCTGGA GAGAGGCTGC TC - #CGTGACCC     240    - CACCATGTCC TCTCCTACCA CGAGTTCCCT GGATACCCCC CTGCCTGGAA AT - #GGCCCCCC     300    - TCAGCCTGGC GCCCCTTCTT CTTCACCCAC TGTAAAGGAG GAGGGTCCGG AG - #CCGTGGCC     360    - CGGGGGTCCG GACCCTGATG TCCCAGGCAC TGATGAGGCC AGCTCAGCCT GC - #AGCACAGA     420    - CTGGGTCATC CCAGATCCCG AAGAGGAACC AGAGCGCAAG CGAAAGAAGG GC - #CCAGCCCC     480    - GAAGATGCTG GGCCACGAGC TTTGCCGTGT CTGTGGGGAC AAGGCCTCCG GC - #TTCCACTA     540    - CAACGTGCTC AGCTGCGAAG GCTGCAAGGG CTTCTTCCGG CGCAGTGTGG TC - #CGTGGTGG     600    - GGCCAGGCGC TATGCCTGCC GGGGTGGCGG AACCTGCCAG ATGGACGCTT TC - #ATGCGGCG     660    - CAAGTGCCAG CAGTGCCGGC TGCGCAAGTG CAAGGAGGCA GGGATGAGGG AG - #CAGTGCGT     720    - CCTTTCTGAA GAACAGATCC GGAAGAAGAA GATTCGGAAA CAGCAGCAGC AG - #GAGTCACA     780    - GTCACAGTCG CAGTCACCTG TGGGGCCGCA GGGCAGCAGC AGCTCAGCCT CT - #GGGCCTGG     840    - GGCTTCCCCT GGTGGATCTG AGGCAGGCAG CCAGGGCTCC GGGGAAGGCG AG - #GGTGTCCA     900    - GCTAACAGCG GCTCAAGAAC TAATGATCCA GCAGTTGGTG GCGGCCCAAC TG - #CAGTGCAA     960    - CAAACGCTCC TTCTCCGACC AGCCCAAAGT CACGCCCTGG CCCCTGGGCG CA - #GACCCCCA    1020    - GTCCCGAGAT GCCCGCCAGC AACGCTTTGC CCACTTCACG GAGCTGGCCA TC - #ATCTCAGT    1080    - CCAGGAGATC GTGGACTTCG CTAAGCAAGT GCCTGGTTTC CTGCAGCTGG GC - #CGGGAGGA    1140    - CCAGATCGCC CTCCTGAAGG CATCCACTAT CGAGATCATG CTGCTAGAGA CA - #GCCAGGCG    1200    - CTACAACCAC GAGACAGAGT GTATCACCTT CTTGAAGGAC TTCACCTACA GC - #AAGGACGA    1260    - CTTCCACCGT GCAGGCCTGC AGGTGGAGTT CATCAACCCC ATCTTCGAGT TC - #TCGCGGGC    1320    - CATGCGGCGG CTGGGCCTGG ACGACGCTGA GTACGCCCTG CTCATCGCCA TC - #AACATCTT    1380    - CTCGGCCGAC CGGCCCAACG TGCAGGAGCC GGGCCGCGTG GAGGCGTTGC AG - #CAGCCCTA    1440    - CGTGGAGGCG CTGCTGTCCT ACACGCGCAT CAAGAGGCCG CAGGACCAGC TG - #CGCTTCCC    1500    - GCGCATGCTC ATGAAGCTGG TGAGCCTGCG CACGCTGAGC TCTGTGCACT CG - #GAGCAGGT    1560    - CTTCGCCTTG CGGCTCCAGG ACAAGAAGCT GCCGCCTCTG CTGTCGGAGA TC - #TGGGACGT    1620    - CCACGAGTGA GGGGCTGGCC ACCCAGCCCC ACAGCCTTGC CTGACCACCC TC - #CAGCAGAT    1680    - AGACGCCGGC ACCCCTTCCT CTTCCTAGGG TGGAAGGGGC CCTGGGCGAG CC - #TGTAGACC    1740    - TATCGGCTCT CATCCCTTGG GATAAGCCCC AGTCCAGGTC CAGGAGGCTC CC - #TCCCTGCC    1800    - CAGCGAGTCT TCCAGAAGGG GTGAAAGGGT TGCAGGTCCC GACCACTGAC CC - #TTCCCGGC    1860    - TGCCCTCCCT CCCCAGCTTA CACCTCAAGC CCAGCACGCA GCGTACCTTG AA - #CAGAGGGA    1920    - GGGGAGGACC CATGGCTCTC CCCCCCTAGC CCGGGAGACC AGGGGCCTTC CT - #CTTCCTCT    1980    #            2030AAAATA AAAACAGAAA AAAAAAAAAA AAAAAAAAAA    - (2) INFORMATION FOR SEQ ID NO:2:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 461 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -    (iii) HYPOTHETICAL: NO    -     (iv) ANTI-SENSE: NO    #ID NO:2: (xi) SEQUENCE DESCRIPTION: SEQ    -      Met Ser Ser Pro Thr Thr Ser Ser - # Leu Asp Thr Pro Leu Pro Gly    Asn    #   15    -      Gly Pro Pro Gln Pro Gly Ala Pro - # Ser Ser Ser Pro Thr Val Lys    Glu    #                 30    -      Glu Gly Pro Glu Pro Trp Pro Gly - # Gly Pro Asp Pro Asp Val Pro    Gly    #             45    -      Thr Asp Glu Ala Ser Ser Ala Cys - # Ser Thr Asp Trp Val Ile Pro    Asp    #         60    -      Pro Glu Glu Glu Pro Glu Arg Lys - # Arg Lys Lys Gly Pro Ala Pro    Lys    #     80    -      Met Leu Gly His Glu Leu Cys Arg - # Val Cys Gly Asp Lys Ala Ser    Gly    #   95    -      Phe His Tyr Asn Val Leu Ser Cys - # Glu Gly Cys Lys Gly Phe Phe    Arg    #                110    -      Arg Ser Val Val Arg Gly Gly Ala - # Arg Arg Tyr Ala Cys Arg Gly    Gly    #            125    -      Gly Thr Cys Gln Met Asp Ala Phe - # Met Arg Arg Lys Cys Gln Gln    Cys    #        140    -      Arg Leu Arg Lys Cys Lys Glu Ala - # Gly Met Arg Glu Gln Cys Val    Leu    #    160    -      Ser Glu Glu Gln Ile Arg Lys Lys - # Lys Ile Arg Lys Gln Gln Gln    Gln    #   175    -      Glu Ser Gln Ser Gln Ser Gln Ser - # Pro Val Gly Pro Gln Gly Ser    Ser    #                190    -      Ser Ser Ala Ser Gly Pro Gly Ala - # Ser Pro Gly Gly Ser Glu Ala    Gly    #            205    -      Ser Gln Gly Ser Gly Glu Gly Glu - # Gly Val Gln Leu Thr Ala Ala    Gln    #        220    -      Glu Leu Met Ile Gln Gln Leu Val - # Ala Ala Gln Leu Gln Cys Asn    Lys    #    240    -      Arg Ser Phe Ser Asp Gln Pro Lys - # Val Thr Pro Trp Pro Leu Gly    Ala    #   255    -      Asp Pro Gln Ser Arg Asp Ala Arg - # Gln Gln Arg Phe Ala His Phe    Thr    #                270    -      Glu Leu Ala Ile Ile Ser Val Gln - # Glu Ile Val Asp Phe Ala Lys    Gln    #            285    -      Val Pro Gly Phe Leu Gln Leu Gly - # Arg Glu Asp Gln Ile Ala Leu    Leu    #        300    -      Lys Ala Ser Thr Ile Glu Ile Met - # Leu Leu Glu Thr Ala Arg Arg    Tyr    #    320    -      Asn His Glu Thr Glu Cys Ile Thr - # Phe Leu Lys Asp Phe Thr Tyr    Ser    #   335    -      Lys Asp Asp Phe His Arg Ala Gly - # Leu Gln Val Glu Phe Ile Asn    Pro    #                350    -      Ile Phe Glu Phe Ser Arg Ala Met - # Arg Arg Leu Gly Leu Asp Asp    Ala    #            365    -      Glu Tyr Ala Leu Leu Ile Ala Ile - # Asn Ile Phe Ser Ala Asp Arg    Pro    #        380    -      Asn Val Gln Glu Pro Gly Arg Val - # Glu Ala Leu Gln Gln Pro Tyr    Val    #    400    -      Glu Ala Leu Leu Ser Tyr Thr Arg - # Ile Lys Arg Pro Gln Asp Gln    Leu    #   415    -      Arg Phe Pro Arg Met Leu Met Lys - # Leu Val Ser Leu Arg Thr Leu    Ser    #                430    -      Ser Val His Ser Glu Gln Val Phe - # Ala Leu Arg Leu Gln Asp Lys    Lys    #            445    -      Leu Pro Pro Leu Leu Ser Glu Ile - # Trp Asp Val His Glu    #        460    - (2) INFORMATION FOR SEQ ID NO:3:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 25 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    #ID NO:3: (xi) SEQUENCE DESCRIPTION: SEQ    #               25 TGCA ARGSC    - (2) INFORMATION FOR SEQ ID NO:4:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 31 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    #ID NO:4: (xi) SEQUENCE DESCRIPTION: SEQ    #          31      TGCA ARGSCTTCTT C    - (2) INFORMATION FOR SEQ ID NO:5:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 31 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    #ID NO:5: (xi) SEQUENCE DESCRIPTION: SEQ    -      Gly Gly Ala Ala Thr Thr Cys Arg - # Ala Ala Asn Cys Cys Asn Gly    Gly    #   15    -      Asn Ala Asn Asn Asn Asn Tyr Thr - # Thr Asn Gly Cys Arg Ala Ala    #                 30    __________________________________________________________________________

What is claimed is:
 1. A human steroid receptor NER, the receptor beingfree of other human receptor proteins and comprising the amino acidsequence of SEQ ID NO:
 2. 2. A DNA sequence encoding human steroidreceptor NER, the sequence being free of other human DNA sequences andcomprising the sequence of SEQ ID NO:1.
 3. A DNA sequence comprisingcodons encoding the protein having the amino acid sequence of SEQ IDNO:2.
 4. An expression construct which comprises:(a) a mammalian cellvector, and (b) a nucleotide sequence encoding human steroid receptorNER protein having the amino acid sequence of SEQ ID NO:
 2. 5. COS cellstransfected with the expression construct of claim
 4. 6. The expressionconstruct of claim 4 wherein the nucleotide sequence comprises SEQ IDNO:
 1. 7. COS cells transfected with the expression construct of claim6.